Studies of the regulation of human cytotoxic T lymphocyte (CTL) development were continued. Alloantigen specific CTL clones were developed to further studies of the roles of cytokines in the development and regulation of CTL activity. While some clones were dependent upon IL-4 and/or IL-7 for the development of cytotoxicity early in their growth, after repeated refeedings and restimulations, they developed activity independent of these cytokines. Serine esterase levels confirm these findings. The clones described above were periodically restimulated with irradiated, allogeneic lymphocytes, a potential source of differentiating cytokines, and cell free and fresh lymphocyte free stimulating systems were established. In the cell free system, microtiter wells were coated with anti-CD3 antibodies and lymphocytes cultured therein in the presence of IL-2 and other cytokines. This system has confirmed the importance of IL- 7 in CTL development previously described in our lab and will allow genetic and cellular level investigation of this phenomenon in relatively pure CTL precursor populations. A fresh lymphocyte-free CTL precursor stimulating system with a very high cloning efficiency for CD8+ T cells (approximately 50%) was developed. OKT3 hybridoma cells were used as feeders and stimulators. Clones developed in this manner showed various patterns of cytokine dependence. As with the alloantigen-specific clones, these clones tended to lose cytokine dependence over time. This system is being used to study CTL precursor frequency under various conditions using limiting dilution analysis. Studies to date have confirmed our previously reported findings that IL-4 can augment CTL development if added after initial activation but is suppressive if added earlier. The ability to study these effects on the clonal level through limiting dilution analysis allows more detailed inspection of the mechanism of the suppressive effect including determination of the target cell population of the effect.